Project Summary The Paramyxoviridae family of viruses includes many pathogens that negatively impact global human health. Human parainfluenza viruses (HPIVs) and respiratory syncytial virus (RSV), are a leading cause of childhood croup, bronchiolitis, and pneumonia cases, leading to tens of thousands of hospitalizations and deaths annually in the U.S.14 Hendra virus (HeV) and Nipah virus (NiV) are emerging infectious agents with high fatality rates due to infection-induced encephalitis.16,17 Despite the demand for therapeutics to combat these pathogens, few effective treatment options are clinically available; existing treatments are somewhat limited by concerns of toxicity or poor efficacy. For this reason, viral fusion has emerged as a compelling target for the development of novel antiviral therapeutics. Paramyxoviral fusion is mediated by a viral fusion (F) protein, which when activated, forms an elongated transient intermediate and exposes two conserved heptad repeat regions (CHR and NHR). The CHR and NHR segments then come together to form a six-helix-bundle (6HB) structure, thermodynamically driving fusion. Segments of paramyxoviral CHR domains from each virus have been shown to bind the transient intermediate of that virus and inhibit viral fusion.9 However, peptides (e.g., 1 and 2; See Fig. 2 of Research Strategy for corresponding sequences) derived from the CHR of HPIV3, have been shown to exhibit activity against several members of the paramyxoviral family.10,11 Despite the conserved fusion mechanism, paramyxoviral F proteins possess distinct primary sequences; therefore, the ability of a single peptide to disrupt the fusion machinery within multiple viruses is highly puzzling. The proposed research focuses on (1) structural characterization of the interactions between broad spectrum antiviral peptides and viral fusion proteins and (2) the design, synthesis, and evaluation of peptidomimetic inhibitors with unnatural amide backbones to mimic those interactions. I will use co-crystallization techniques to pursue x-ray structures of 1 and 2 bound to their viral targets in HPIV3, RSV, NiV, and HeV F. Already, these efforts have led to crystal structures of 1 bound to PIV3 NHR and 2 bound to RSV NHR, which reveal atomic-level insights that will guide future inhibitor development. I will then use structure-guided design principles to incorporate ?- and g-amino acid residues into the sequences of 1 and 2 to create foldamers that structurally and functionally mimic the diverse secondary structural elements observed in preliminary structures of the ?-peptides. Peptidomimetics will be evaluated for binding affinity to paramyxoviral NHR domains using competitive fluorescence polarization and thermal shift assays. With the guidance of Prof. Anne Moscona, a molecular virologist at Columbia University (See Letter of Collaboration), lead peptidomimetics will be evaluated for inhibition of fusion mediated by PIV3-, RSV-, and NiV F and efficacy against infection by wild-type PIV3 and RSV, or pseudotyped NiV.